The overall goal of Project 3 is to discover novel anticancer drugs based on designing small synthetic molecules that inhibit the chymotrypsin-like activity of the proteasome. The proteasome is a multi-protease complex that is responsible for degrading proteins such as the tumor suppressor p53, the cyclin-dependent kinase inhibitors p27 and p21 and the proapoptotic proteins Bax and 1KB. Many tumors are dependent on the proteasome to evade programmed cell death (apoptosis) and survive. The majority of human cancers have low levels of substrates of the proteasome such as p53, p27 and Bax and this has been associated with tumor aggressiveness, poor prognosis, resistance to chemotherapy and shortened patient survival time. The hypothesis upon which Project 3 is based is that proteasome selective inhibitors will block proteasome mediated degradation of pro-apoptotic proteins, andwill suppress tumor growth by inducing apoptosis. To test this hypothesis the following specific aims are proposed: 1) To use high throughput screening (HTS) and chemical library design to identify chymotrypsin-like selective proteasome inhibitors. 2) To evaluate the potency and selectivity of the compounds identified in Specific Aim 1 to inhibit the proteasome chymotrypsin-like activity, and to correlate the inhibitors'potency to their ability to induce apoptosis and cell cycle arrest, and to inhibit malignant transformation. To determine if proteasome inhibitors are selective for cancer cells over normal cells. 3) To determine whether proteasome inhibitor- induced cancer cell apoptosis is mediated by accumulation of pro-apoptotic protein. 4) To determine if suppression of the proteasome chymotrypsin-like activity is required for BclXL/Bax (Project 4), and mdm2/p53 arid/or mdmx/p53 (Project 1) disrupters to induce apoptosis. 5) To evaluate the ability of proteasome inhibitors, that selectively inhibit chymotrypsin-like activity, to induce apoptosis and inhibit tumor growth in animal models using human tumor xenografts. To carry out pharmacodynamic, combination therapy and toxicity studies. The studies proposed will provide the chemical probes that will allow us to enhance our understanding of how the proteasome regulates aberrant signal transduction pathways such as IKB/NFKB, mdm2/p53 and/or mdmx/p53 and BclXL/Bax that are critical to tumor survival/death. Ultimately, the studies will lead to the discovery of chymotrypsin-like proteasome inhibitors with proapoptotic and antitumor activities, and will broaden the spectrum of human tumors that can be successfully treated.